Engineering the Expression and Characterization of Two Novel Laccase Isoenzymes from Coprinus comatus in Pichia pastoris by Fusing an Additional Ten Amino Acids Tag at N-Terminus
Ding S (2014) Engineering the Expression and Characterization of Two Novel Laccase Isoenzymes from Coprinus comatus
in Pichia pastoris by Fusing an Additional Ten Amino Acids Tag at N-Terminus. PLoS ONE 9(4): e93912. doi:10.1371/journal.pone.0093912
Engineering the Expression and Characterization of Two Novel Laccase Isoenzymes from Coprinus comatus in Pichia pastori s by Fusing an Additional Ten Amino Acids Tag at N-Terminus
Chunjuan Gu 0
Fei Zheng 0
Liangkun Long 0
Jing Wang 0
Shaojun Ding 0
Ligia O. Martins, Universidade Nova de Lisboa, Portugal
0 Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University , Nanjing, Jiangsu , China
The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named lac3 and lac4, encoding proteins of 547 and 532amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete Coprinus comatus. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in Pichia pastoris by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser) tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest Km and highest kcat/Km value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of kcat/Km) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes.
Funding: This work was supported by the National Natural Science Foundation of China (NSFC) (Grant No. 30871987) and the Project Funded by the Priority
Academic Program Development of Jiangsu Higher Education Institutions. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Laccases [benzenediol: oxygen oxidoreductases (EC22.214.171.124)]
are copper-containing enzymes capable of oxidizing a broad
spectrum of phenolic compounds and non-phenolic substrates
using molecular oxygen as the electron acceptor. In fungi, laccases
probably play critical roles in several physiological functions, such
as morphogenesis, fungal plant-pathogen/host interaction,
degradation of lignocellulosic material, and pigment production .
Due to their great versatility and broad substrate specificity,
laccases can be used for several industrial applications, such as
pulp bleaching in paper industry, textile dye decolorization and
detoxification of environmental pollutants . Laccases might also
be useful in synthetic chemistry . For instance, laccases have
been used to synthesize dyes  and products of pharmaceutical
importance, such as the anticancer drug mitomycins . The
practical applications of laccases in bio-technology would require
the suitable laccase for each purpose and economical production
of great quantities of pure protein.
In many fungal species, laccases occur as groups of isoenzymes
encoded by gene families . For example, in the Coprinopsis cinerea
genome, 17 nonallelic laccase genes were identified and at least 9
of these members are translated into functional laccase products
. Laccase isoenzymes had a highly similar primary structure but
differences in expression level and physico-chemical
characteristics, thus making difficult to purify individual isoenzymes from
fungal cultures for analysis and application. Another approach is
the heterologous expression of laccase isoenzymes in a suitable
host. Laccase genes have been heterologously expressed in the
yeasts Saccharomyces cerevisiae and Pichia pastoris [8,9], or filamentous
fungi, such as Trichoderma reesei , and Aspergillus spp. [11,12].
However, notoriously laccases, like other ligninolytic enzymes are
relatively difficult to express heterologously in an active form in
host contrary to other enzymes. So far, despite the fact that an ever
increasing number of laccase gene families has been identified
from various fungi by traditional cloning and genome sequencing,
only limited number of laccase isoenzymes restricted to few fungi
such as Pleurotus ostreatus, Trametes versicolor and Lentinula edodes have
been biochemically characterized . Accordingly, further
understanding of precise physiological/biochemical roles of
individual laccase isoenzymes in different fungi is necessary for
fundamental and application purposes.
59-CAY TGG CAY GGN TTY TTY CA-39
59-G RCT GTG GTA CCA GAA NGT NCC-39
In the degenerate primers the following abbreviations were used Y = C, T; N = A, G, C, T; M = C, A; R = A, G; V = G, A, C.
Coprinus comatus is widely cultivated in many countries as a
delicious and highly nutritious edible. The edible mushroom C.
comatus produced multiple extracellular laccase isozymes, among
them, one laccase isoenzyme Lac1 was characterized and
exhibited a promising potential in the degradation of some
recalcitrant synthetic dyes . The complexity of laccases along
with their role in biotechnological applications led us to further
investigate other isoenzymes in laccase family of C. comatus. In the
current study, we successfully expressed two novel laccase
isoenzymes in P. pastoris by fusing an additional 10 amino acids
tag at N-terminus. The biochemical properties and decolorization
potentials of two laccase isoenzymes were also compared. The aim
of this work was to expand our knowledge of individual laccase
isoenzymes in fungi, and in order to identify the new laccase
isoenzymes as the candidates for industrial applications.
Materials and Methods
Fungal strain, media, and culture conditions
C. comatus was provided by the Institute of Edible Fungi,
Shanghai Academy of Agricultural Sciences, Shanghai, China,
and maintained on potato dextrose agar at 4uC with periodic
transfer. Escherichia coli DH5a was used as the host for recombinant
plasmids. pGEM-T vector (Promega) was used to subclone DNA
fragments for sequencing. The vector pPICZaB (Invitrogen) was
used for gene expression in P. pastoris. All media and protocols for
Pichia were according to the Pichia expression manual
(Invitrogen). For extraction of RNA, the fungus was grown in stationary
250 ml Erlenmeyer containing 50 ml basal medium with 1 mM
caffeic acid at 25uC for 22 days as described previously . Basal
medium contained (per liter): 1.0 g KH2PO4, 0.4 g K2HPO4,
0.5 g MgSO4?H2O, 0.013 g CaCl2?2H2O, 0.1 g yeast extract,
0.5 g NH4NO3, 3.0 g asparagine and 2 ml Tween 80. After
autoclaving and cooling to room temperature, 2.5 mg/l thiamine
and 1 ml/l of a trace-elements solution consisting of (per liter):
4.8 g FeC6H5O7?5H2O, 2.64 g ZnSO4?4H2O, 2.0 g
MnCl2?4H2O, 0.4 g CoCl2?6H2O and 0.4 g CuSO4?5H2O was
added. Four agar discs (1 cm diameter), cut from the growing edge
of a 7-day PDA culture, were used to inoculate each flask.
Cloning of laccase isoenzymes lac3 and lac4
The genomic DNA was extracted from C. comatus with the
method described by Hoshida et al. . Total RNA was isolated
from 22-day-old mycelia using a TRizol reagent (Invitrogen)
according to the manufacturer instructions and used for cDNA
synthesis. Degenerate primers LacCu1 and LacCu2 (Table 1) were
designed according to the conserved sequences of the
copperbinding regions I (HWHGFFQ) and II (GTFWYHS) in most
fungal laccases, respectively. PCR was carried out using Taq DNA
polymerase (Takara), and the genomic DNA as the template. The
PCR reaction programme was initiated at 94uC for 5 min,
followed by 30 cycles of 94uC for 30 s, 55uC for 30 s and 72uC for
2 min, and a final extension at 72uC for 10 min. The 200 bp PCR
products were cloned into the pGEM-T vector and 12 randomly
selected clones were sequenced. The nucleotide sequences of the
12 clones were classified into 3 groups. Among them, one was
identical with the previously reported lac2, two were new laccase
isoenzymes, designed as lac3 and lac4, respectively. The 59-end
cDNA fragments were amplified by RACE-PCR using the
SMART RACE cDNA Amplification Kit (Clontech) and primers
Lac3 GSP1 and Lac4 GSP1 for lac3 and lac4, respectively. The
fragments were subcloned into pGEM-T vector and sequenced.
The full-length cDNAs of lac3 and lac4 were then generated by
39RACE using the gene-specific primers Lac3GSP2 and Lac4GSP2
designed from the sequence of the extreme 59-cDNA ends of lac3
and lac4 respectively, and sequenced as above. Phylogenetic
analyses were performed using the MEGA version 5.0 software
. Sequences were aligned globally using the Clustal W
program in MEGA. Trees were constructed by the
neighborjoining method with a Poisson correction model.
Construction of expression plasmids
Six different expression plasmids were constructed in order to
functionally express lac3 and lac4 in P. pastoris, (Fig. 1). For the
constructs with mature laccase genes, the lac3 and lac4 genes were
PCR-amplified using primer pairs Lac3F/Lac3R, and Lac4F/
Lac4R respectively. After double digestion with EcoR I and Not I,
the fragments were ligated with the same digested vector
pPICZaB in frame with the C-terminal myc epitope and the
polyhistidine tag to yield the constructs, pPICZaB-Lac3 and
pPICZaB-Lac4. For expression of laccase genes containing an
additional N-terminal 10 amino acids tag, the expression vector
pPICZaB-10AA was generated by PCR using primer pair
pPICZaB-10AAF/pPICZaB-10AAR and template pPICZaB. In
which the phenylalanine and proline rich motif
Thr-Pro-Phe-ProPro-Phe-Asn-Thr-Asn-Ser (TPFPPFNTNS), derived from the first
N-terminal 10 amino acids residues of the mature xylanase (XynII)
in V. volvacea , was fused with pPICZaB at EcoRI site. Then
the lac3 and lac4 genes were PCR-amplified using primer pairs
Lac310AAF/Lac310AAR and Lac410AAF/Lac410AAR
respectively. Both of the amplified pPICZaB-10AA and gene products
were flanked by Xba I and Sac II restriction sites at the 59- and
39ends, respectively. After double digestion with XbaI and Sac II,
the fragments were ligated with the same digested pPICZaB-10AA
in frame with the C-terminal myc epitope and the polyhistidine tag
to yield the constructs, pPICZaB-10AALac3 or
pPICZaB10AALac4. For the constructs without Myc tag, the lac3 and
lac4 genes with 66His tag followed by a termination codon at
Cterminus were PCR-amplified using primer pairs Lac310AAF/
Lac3mycR and Lac410AAF/Lac4mycR respectively. The
fragments were inserted into the pPICZaB-10AA after digested with
Xba I and Sac II to yield plasmids pPICZaB-10AALac3-DMyc
and pPICZaB-10AALac4-DMyc. All the cDNA constructs were
verified by DNA sequence analyses. PCR conditions were: one
cycle of 94uC for 5 min, 55uC for 30 s and 72uC for 3 min; 30
cycles of 94uC for 30 s, 55uC for 30 s and 72uC for 3 min followed
by a final extension at 72uC for 5 min.
Expression of laccase isoenzymes lac3 and lac4
All the expression plasmids were linearized with SacI and
transformed into P. pastoris KM71H competent cells by
electroporation with a Genepulser II apparatus (Bio-Rad, Hercules, CA).
Transformants containing the lac3 and lac4 cDNA were selected
on yeast extract-peptone-dextrose (YPD) agar plates with 1 M
sorbitol and 100 mg/ml Zeocin (Invitrogen). The candidate
transformants were transferred to minimal methanol (MM) plates
containing 0.2 mM ABTS and 0.1 mM CuSO4.
Laccase-producing transformants were identified by the presence of dark green
color appearance around the colonies after four days growth. The
high expression transformants which showing a deeper color in the
plate were selected for liquid fermentance experiment. The
transformed yeast cells were grown in 250 ml flasks containing
50 ml BMGY medium at 250 rpm and 30uC. After the OD600
value reached 6, cells were harvested by centrifugation, and
resuspended in 15 ml of BMMY medium containing 500 mM
CuSO4 in 250 ml Erlenmeyer flasks and induced for a further 15
days by adding methanol to a final concentration of 0.5% at 24 h
intervals. Every 24 h, 0.5 ml of cultures was sampled from the
flasks and laccase activity was determined after separating yeast
cells by centrifugation.
Enzyme activity assay
The routine assay for laccase activity towards
2,29-azino-bis-[3ethylthiazoline-6-sulfonate] (ABTS) as the substrate was measured
in 1 ml reaction volume containing 1 mM ABTS, 100 mM
sodium acetate buffer (pH 4.5) and 5 ml aliquots of appropriately
diluted enzyme sample. Oxidation of ABTS was monitored by
following the increment in A 420 (e420 = 3.66104 M21cm21) .
One unit of laccase activity was defined as the amount of enzyme
required to oxidize 1 mmol ABTS min21 at 40uC. The activities
towards syringaldazine and 2,6-dimethylphenol were assayed
under same condition as above, oxidation of syringaldazine and
2,6-dimethylphenol was followed by an absorbance increase at
525 nm (e525 = 6.56104 M21.cm21) and 421 nm
(e421 = 5.786104 M21.cm21), respectively. The activity towards
guaiacol was measured in 2.4 ml reaction mixture containing
1 mM guaiacol, oxidation of guaiacol was monitored by an
absorbance increase at 465 nm (e465 = 1.216104 M21.cm21) after
30 min reaction. Protein concentration was determined by a BCA
Protein Assay Kit (Thermo Scientific Pierce) with bovine serum
albumin as standard.
Purification and characterization of recombinant Lac3
Culture supernatants from 500 ml BMMY cultures were
collected by centrifugation (5,000 g for 15 min) and concentrated
,20-fold with the Pellicon ultrafiltration system (Millipore) using a
10 kDa molecular mass cut-off membrane. The concentrated
enzyme solution was dialyzed overnight against 20 mM TrisHCl
(pH 8.0) and any remaining precipitate was removed by
centrifugation at 10,000 g for 30 min. The supernatant was applied to a
DEAE Sepharose CL-6B (1.6650 cm, Dingguo, Beijing, China)
pre-equilibrated with the same buffer. After washed with same
buffer to remove unbound proteins, the bound laccases were
subsequently eluted from the column with a linear salt gradient (0
1.0 M NaCl) in the same buffer. The active fractions were pooled,
concentrated to,5 ml by ultrafiltration and further purified by
fast protein liquid chromatography (FPLC) gel filtration on a
Superdex 75 HR column (Tricorn-10/600 47.5 ml, GE
Healthcare) in the same buffer (pH 7.5) using an AKTA Purifier (GE
Healthcare). The combined active fractions were concentrated to
,3 ml by ultrafiltration as described above, and stored at 220uC
for further use. Enzyme homogeneity and the molecular weight of
purified Lac3 and Lac4 were estimated using sodium dodecyl
sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (10% w/
v) and the protein molecular weight markers (TaKaRa) as a
reference under reducing conditions. Optimal pH and
temperature values were determined using the ABTS, guaiacol,
2,6dimethylphenol and syringaldazine (each at 1 mM) as substrates
over the ranges pH 1.09.0 (100 mM KClHCl buffer pH 1.0
2.0, universal buffer: 50 mM H3PO4, 50 mM CH3 COOH,
50 mM H3BO3, pH 2.09.0 adjusted with 0.2 M NaOH at 25uC)
and 3080uC, respectively. The thermal stability was determined
by measuring the residual activities with substrate ABTS after
preincubation of the enzymes at 30uC to 70uC for 060 min. The
kinetic constants (Vmax and Km) were respectively determined by
measuring the rates of substrates oxidation using substrate
concentration ranges of 0.31.0 mM, 0.061 mM, 0.31.0 mM,
and 0.080.6 mM for ABTS, guaiacol, 2,6-dimethylphenol and
syringaldazine at their optimal temperature and pH value. The
pH values for substrate specificity and kinetic constants measures
were 3.0, 5.0, 5.5, and 5.0 for Lac3, and 3.0, 6.0, 5.5 and 6.0 for
Lac4 for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine,
respectively. The Vmax and Km values were calculated by
Graphpad Prism 5.0 software (http://www.graphpad.com/
prism/) using nonlinear regression. The effect of various metal
ions (each at 1 mM) and inhibitors (EDTA, SDS, each at 5
100 mM; DTT, 0.0011 mM) on Lac3 and Lac4 activity was
assessed by pre-incubating the purified enzyme (0.2 IU/ml) with
different metal ions and inhibitors at 4uC for 30 min. The residual
activity was determined using ABTS as a substrate under the
standard assay conditions.
Deglycosylation of recombinant Lac3 and Lac4
Purified recombinant Lac3 and Lac4 were treated with
endoglycosidase H (endo- N-acetylglucosaminidase H of
Streptomyces plicatus; NEB) according to the manufacturers protocol. Lac3
or Lac4 (2 mg each) in 16 glycoprotein denaturing buffer was
boiled for 10 min. After cooling, 1 ml of endoglycosidase H was
added and the sample was incubated at 37uC overnight. The
product of endoglycosidase H treatment was analyzed by
Thirteen different dyes were used for this study. The reaction
mixture (2 ml) contained 100 mM acetate buffer pH 4.5,
individual dye (each 50 mg/l in final concentration), and 0.5 IU laccase
with or without a redox mediator 1-hydrox-ybenzotriazol (HBT,
final concentration 0.1 mg/ml). The reaction was initiated by the
addition of laccase and incubated at 40uC for 12 h. For synergistic
decolorization by two laccases, Lac3 and Lac4 (0.25 IU each) were
added in reaction mixture. Decolorization was determined by
monitoring the decrease in absorbance at the peak of maximum
visible absorbance and expressed as percentage of decolorization.
Decolorization was defined as: Decolorization (%) = 1006(Ao
At)/Ao. Where Ao is the absorbance of the reaction mixture before
incubation with the enzyme and At is absorbance after incubation.
The heat-denatured laccase solutions were used as controls and
the blanks contained all components of the reaction mixture
except the dyes.
identities respectively. A phylogenetic analysis of protein sequences
revealed that lac3 and lac4 belonged to the same phylogenetic
Nucleotide sequence accession numbers
The nucleotide sequences of lac3 and lac4 were deposited in the
European Nucleotide Archive (ENA) under accession numbers
HG764548 (lac3) and HG764549 (lac4) respectively.
Cloning and sequence analysis of lac3 and lac4
Two cDNAs encoding new laccase isoenzymes Lac3 and Lac4
in C. comatus were cloned in this study. As predicted from the
fulllength cDNA clones, lac3 and lac4 encode 547- and 532-aa-long
polypeptides with a putative signal peptide of 28 or 16 amino
acids, respectively. They show 70% amino acid pairwise identity
(Fig. 2), and each contains 3 potential N-glycosylation sites. The
calculated pI values of predicted mature Lac3 and Lac4 are 5.16
and 5.8, respectively. The deduced amino acid sequences of lac3
and lac4 were compared with the sequence of other laccases
available in the GenBank database. Lac3 was most closely related
to Laccaria bicolor laccase (XP_001881441.1) and Coprinopsis cinerea
laccase 4 (XP_001829763.2) with a 57% identity, followed by
Lentinula edodes laccase (AET86511.1), Coprinopsis cinerea laccase 8
(AAR01249.1) and laccase 1 with 55%, 53% and 53% identities
respectively. Whereas lac4 exhibited the maximum identity (58%)
with Laccaria bicolor laccase (XP_001881441.1), followed by
Coprinopsis cinerea laccase 4(XP_001829763.2), laccase 8
(AAR01249.1) and laccase 1 (AAS38574.1), and Cyathus bulleri
laccase (ABW75771.2) with 56%, 56%, 55%, 54%, and 54%
Heterologous expression of lac3 and lac4 in Pichia
To express the lac3 and lac4 in P. pastoris, six different expression
plasmids were constructed under the control of the tightly
regulated AOX1 promoter. By the plate detection, the dark green
zones appeared around the transformants containing constructs
pPICZaB-10AALac3 or pPICZaB-10AALac4, carrying the fusion
laccase genes with an additional N-terminal 10 aa tag in frame
with the C-terminal myc epitope and the polyhistidine tag (Fig. 3A
and 3B). The results implied that bioactive Lac3 and Lac4 were
expressed and secreted into the extracellular medium. On the
contrary, no dark green zones appeared around the transformants
containing constructs pPICZaB-Lac3, pPICZaB-Lac4,
pPICZaB10AALac3-DMyc or pPICZaB-10AALac4-DMyc, demonstrating
the essential role of the additional 10AA tag at N-terminus and
myc epitope at C-terminus in lac3 and lac4 expressions in P.
pastoris. The laccase-positive transformants with constructs
pPICZaB-10AALac3 or pPICZaB-10AALac4, as well as the
laccasenegative transformants, were then fermented in BMMY liquid
medium at 30uC and induced by addition of 0.5% (v/v) methanol
daily. After 14-days growth, the laccase activities reached 689 and
1465 IU/l for Lac3 and Lac4 respectively (Fig. 3C). No
extracellular laccase activity was detected in culture supernatants
of the laccase-negative transformants containing pPICZaB-Lac3,
pPICZaB-Lac4, pPICZaB-10AALac3-DMyc or
pPICZaB-10AALac4-DMyc respectively. Lac2 gene, which could be not expressed
in P. pastoris, was used to assess whether the N-terminal 10 aa
tag is effective for other laccases expression. The green zone
appeared around the transformant containing plasmid
pPICZaB10AALac2, carrying the fusion lac2 gene with the N-terminal 10
aa tag in frame with the C-terminal myc epitope and the
polyhistidine tag (Fig. S2A). The laccase activity reached 60.1 U/l
after 14-days growth at 30uC (Fig. S2B).
Purification and characterization of recombinant Lac3
The recombinant Lac3 and Lac4 were purified by
chromatography with two steps procedure. SDS-PAGE analysis revealed that
the molecular masses of purified recombinant Lac3 and Lac4 were
about 75 kDa and 70 kDa, which was higher than the predicted
masses of 54 and 53 kDa, respectively (Fig. 4A and 4B). After
treatment with endoglycosidase H (endoH), the molecular masses
of Lac3 and Lac4 were reduced to 55 kDa and 52 kDa
respectively (Fig. 4A and 4B), indicating the recombinant laccases
were glycosylated. The optimal pH values of Lac3 or Lac4 were
3.0 for ABTS, 5.5 for syringaldazine, 5.0 or 6.0 for guaiacol, and
5.0 or 6.0 for 2,6-dimethylphenol, respectively (Fig. 5A and 5B).
Meanwhile, the optimal temperature values of Lac3 or Lac4 were
65uC or 60uC for ABTS, 60uC or 65uC for syringaldazine, 50uC
or 60uC for guaiacol, and 60uC or 65uC for 2,6-dimethylphenol,
respectively (Fig. 5C and 5D). The recombinant laccases were
stable at temperatures below 50uC (Fig. 5E and 5F), and were very
sensitive to DTT but not to EDTA and SDS (Table 2). Lac3 and
Lac4 showed high resistant to SDS, and retained 31.86% and
43.08% activity in the presence of 100 mM SDS, respectively. The
metal ions such as Ca2+, Mg2+, Co2+, Zn2+, Mn2+ and Cu2+ had
slight or even no effect on the activities of Lac3 and Lac4 (Table 2).
Kinetic parameters of the laccases were determined by using
ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine as
substrates and summarized in table 3. Remarkably, two laccases
demonstrated different substrate affinities and turnover rates (kcat).
Lac3 had significantly lower Km and higher kcat/Km values than
Lac4 towards ABTS, syringaldazine and 2,6-dimethylphenol.
Dye decolorization by recombinant Lac3 and Lac4
Thirteen synthetic dyes including anthraquinone, azo and
triphenylmethane dyes were used to evaluate the decolorization
ability of the recombinant Lac3 and Lac4 with or without HBT.
Among the three types of dyes, the recombinant Lac3 and Lac4
showed higher decolorization efficiency for anthraquinone than
triphenylmethane and azo dyes in the absence of HBT (Fig. 6A).
In the presence of HBT, the decolorization efficiencies of Lac3
respectively increased to 95.34%, 91.7%, and 96.33% for
Remazol brilliant blue R, Reactive dark blue KR and Malachite
green after 12 h of incubation, while the corresponding values by
Lac4 were 95.66%, 89.12% and 93.96% respectively (Fig. 6B).
The other dyes were decolorized to different extend within 12 h as
revealed in Fig. 6. The broad decolorization specificity of Lac3
and Lac4 rendered their great potentials in industrial applications,
such as degradation of dyes from textile effluents. It was interesting
that Lac3 showed higher decolorization ability than Lac4 towards
most of dyes with except of Remazol brilliant blue R and
Bromophenol blue. The mild synergistic decolorization by two
laccases was observed for triphenylmethane dyes, and the
decolorization percentages were enhanced by 5% when mixed
laccases used in reaction comparing with Lac3 or Lac4 alone. But
no synergistic decolorization was detected for anthraquinone and
azo dyes (data not shown).
Two cDNAs encoding novel laccase isoenzymes in C. comatus
were identified in this study. They showed 70% identity but lower
homology with previously cloned Lac1 and Lac2 from C. comatus
and other fungal laccases. At protein level, Lac3 and Lac4 have
50% and 47.2% identities with the Lac1, and 51.9% and 50.6%
identities with the Lac2, respectively. Furthermore, unlike some
high redox potential fungal laccases bearing a Leu or Phe residue
at the amino acid residue 10 aa downstream of the conserved
cysteine respectively, Lac3 and Lac4 have non-coordinating Met
Relative activity (%)
Values are the mean of triplicate determinations and standard deviation is less 5%.
at this position. According to the current literatures, several
structural features may contribute to the redox potential of
laccases, including the amino acids which form the T1 pocket and
the coordination sphere of the T1 copper [22,23]. Lac3 and Lac4
are provisionally grouped into low redox potential classes based on
axial ligand type (Met as axial ligand), as similar with other low
redox potential bilirubin oxidase from ascomycete Myrothecium
verrucaria , laccase from plant Rhus vernicifera  and archaeal
laccase from Haloferax volcanii . The simultaneous presence of
high redox and low redox potential laccases has been reported in
Trametes sp. C30 [27,28].
Various yeast and filamentous fungal species have been used as
hosts to heterologously express laccase genes for their
characterization and application. For example, different laccase isoenzymes
from Trametes versicolor were successfully expressed in yeasts Yarrowia
lipolytica , Pichia methalonica . P. pastoris [13,31] and
Saccharomyces cerevisiae  or filamentous fungi Aspergillus niger
 and Trichoderma reesei . The P. pastoris system may still be
the most frequently used host owing to convenience and speed,
although the expression levels was considerably lower than some
yeasts and filamentous fungi. Over 20 fungal laccases have been
heterologously expressed in P. pastoris for different purposes .
However, the production remained low and even no expression
for some laccases. The inability of yeasts to process the
posttranslation of different laccase genes with the same efficiency may
explain the observed selectivity in expression . There are
several strategies used to increase the expression level of
heterologous proteins in Pichia , such as the use of native
promoters and multiple gene copies, codon optimization, altering
of secretory signal sequences, and optimization of culture
conditions. However, these were not successful in some cases for
fungal laccase expression . In this study, we successfully
expressed two laccase isoenzymes in P. pastoris by fusing the genes
with the N-terminal 10 aa tag from the xylanase of V. volvacea .
The successful expression of lac2 by fusing the additional 10 aa tag
further confirmed the effective of this tag in improving laccase
expression in P. pastoris. The reason for the great increase in the
expression levels was unclear. It has been reported in literuture
that the deletion or added N-terminal amino acids and C-terminal
sequence were beneficial for increasing the protein secretion levels
in P. pastoris [36,37]. It can be safely assumed that the additional
N-terminal 10 aa peptide together with C-terminal myc epitope
may contributes to the correct post-translationally processing of
Lac3 and Lac4 in Pichia. Our data suggested that the additional
phenylalanine and proline rich motif could be useful tag for
increasing heterologous protein expression in yeast.
When laccases were expressed in P. pastoris, the recombinant
laccases were found be hyperglycosylated. However, no reports
show that hyperglycosylation affects the activity of the enzyme
produced . By the molecular mass determination before and
after endo-H deglycosylation, glycosylation patterns of the
recombinant Lac3 and Lac4 was 36.4% and 34.6% respectively,
similar as other fungal laccases expressed in P. pastoris . Two
laccases in general showed low optimum pH for ABTS, but
relative higher for other substrates similar as other fungal laccases
. Lac4 has higher optimal pH values than Lac3 when assayed
with guaiacol and 2,6-dimethylphenol. Lac4 also exhibited higher
optimal temperature than Lac3 for guaiacol, syringaldazine and
2,6-dimethylphenol. Although they demonstrated obvious
differences in substrate affinities and turnover rates (kcat), both laccases
possessed the lowest Km and highest kcat/Km value towards
syringaldazine, followed by ABTS, guaiacol and
2,6-dimethylphenol. From these findings, it is obvious that Lac3 and Lac4 were
clearly distinguishable from the previously cloned Lac1 from C.
comatus  and other well known fungal laccase , but possessed
similar biochemical properties as the low redox potential laccases
from Melanocarpus albomyces  and Trametes sp. C30 Lac2 and
Lac3 [27,28], or archaeal laccase from Haloferax volcanii .
Sodium dodecyl sulphate (SDS) is a strong protein denaturant that
inactivates most laccases even at a low concentration [39,40]. Lac3
and Lac4 displayed remarkably higher SDS resistance than many
of published fungal laccases, thus showing an application potential
in detergent-containing conditions.
Laccase-mediated dye decolorization has been described with
crude or purified forms from many fungal species. The
decolorization rate varied according to the source of enzymes and the
structures of different dyes. The decolorization capability of Lac3
and Lac4 were similar or even higher than many reported fungal
laccases. For example, it was reported that the laccases derived
from P. ostreatus and Ganoderma lucidum could decolorize 70% and
40.7% of malachite green (50 mgl21) after 24 h of incubation
[41,42]. In our study, the decolorization efficiencies reached
89.73% and 83.73% for Lac3 and Lac4 after 12 h of incubation
without redox mediator respectively. Lac3 exhibited higher
decolorization efficiency than Lac4 for 11 out of 13 kinds of the
tested dyes, which may attribute to the relatively higher catalytic
efficiency of Lac3 than Lac4 (in terms of kcat/Km) towards
syringaldazine and ABTS. It was reported that the crude laccases
had more decolorization efficiency than purified laccase probably
due to coexistence of different isoenzymes [43,44]. However, there
was no report to present synergistic interactions between purified
laccase isoenzymes in decolorization previously. In this study, we
observed the synergistic interactions between Lac3 and Lac4 in
dye decolorization, but it is highly dyes dependent.
In conclusion, we successfully expressed two laccase isoenzymes
from C. comatus in P. pastoris by fusing with an additional ten amino
acids tag at N-terminus. The Lac3 and Lac4 showed clearly
different catalytic properties and dye decolorization abilities. So
far, the biological functions of different isoenzymes in fungal
species were still poorly understood. This study reinforced the
laccase diversity in fungi and suggested that these laccase
isoenzymes with a low sequence identity among individual
members have diverse functions in C. comatus. The tolerance of
Lac3 and Lac4 towards extreme conditions, including various
metal ions and high concentration of SDS, as well as wide dye
decolorization ability, demonstrated these laccase isoenzymes
Figure S1 Neighbor-joining tree of the deduced amino
acid sequences of Lac3 and Lac4 and other laccases from
GenBank. The tree is calculated with p-distances using Mega
ver. 5.2, based on a ClustalX alignment. Bootstrap values (1000
replications) higher than 50% are indicated at branchings.
Figure S2 Detection of the recombinant Lac2 laccase
activity on BMMY agar plates containing CuSO4 and
ABTS (A) and the time course of extracellular Lac2
production in BMMY liquid medium (B). A: transformants
containing 1) pPICZaBB-Lac2, and 2) pPICZaB-10AALac2; B:
transformants containing pPICZaB-10AALac2 (filled square), and
pPICZaBB-Lac2 (filled circle).
Conceived and designed the experiments: CG FZ LL JW SD. Performed
the experiments: CG FZ. Analyzed the data: CG FZ LL JW. Contributed
reagents/materials/analysis tools: CG FZ LL JW SD. Wrote the paper:
1. Baldrian P ( 2006 ) Fungal laccases-occurrence and properties . FEMS Microbiol Rev 30 : 215 - 242 .
2. Mayer AM , Staples RC ( 2002 ) Laccase: new functions for an old enzyme . Phytochemistry 60 : 551 - 565 .
3. Kunamneni A , Camarero S , Garca-Burgos C , Plou FJ , Ballesteros A , et al. ( 2008 ) Engineering and applications of fungal laccases for organic synthesis . Microb Cell Fact 7 : 32 .
4. Polak J , Jarosz-Wilkolazka A ( 2012 ) Structure/redox potential relationship of simple organic compounds as potential precursors of dyes for laccase-mediated transformation . Biotechnol Prog 8 : 93 - 102 .
5. Herter S , Michalik D , Mikolasch A , Schmidt M , Wohlgemuth R , et al. ( 2013 ) Laccase-mediated synthesis of 2-methoxy-3-methyl-5-(alkylamino)- and 3- methyl-2,5-bis(alkylamino)-[1,4]-benzoquinones . J Molecular Catalysis B: Enzymatic 90 : 91 - 97
6. Kues U , Ru hl M ( 2011 ) Multiple multicopper oxidase gene families in basidiomycetes - what for? Curr Genomics 12 : 72 - 94 .
7. Kilaru S , Hoegger PJ , Kues U ( 2006 ) The laccase multigene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies . Curr Genet 50 : 45 - 60
8. Bleve G , Lezzi C , Mita G , Rampino P , Perrotta C , et al. ( 2008 ) Molecular cloning and heterologous expression of a laccase gene from Pleurotus eryngii in free and immobilized Saccharomyces cerevisiae cells . Appl Microbiol Biotechnol 79 : 731 - 41 .
9. Hilden K , Makela MR, Lundell T , Kuuskeri J , Chernykh A , et al. ( 2013 ) Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus . Appl Microbiol Biotechnol 97 : 1589 - 1599 .
10. Kiiskinen LL , Kruus K , Bailey M , Ylosmaki E, Siika-Aho M , et al. ( 2004 ) Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme . Microbiology 150 (Pt 9): 3065 - 74 .
11. Rodrguez E , Ruiz-Due ns FJ , Kooistra R , Ram A , Martnez AT , et al. ( 2008 ) Isolation of two laccase genes from the white-rot fungus Pleurotus eryngii and heterologous expression of the pel3 encoded protein . J Biotechnol 134 : 9 - 19 .
12. Yano A , Kikuchi S , Nakagawa Y , Sakamoto Y , Sato T ( 2009 ) Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae . Microbiol Res 164 : 642 - 9 .
13. Koschorreck K , Richter SM , Swierczek A , Beifuss U , Schmid RD , et al. ( 2008 ) Comparative characterization of four laccases from Trametes versicolor concerning phenolic C-C coupling and oxidation of PAHs . Arch Biochem Biophys 474 : 213 - 9 .
14. Piscitelli A , Pezzella C , Giardina P , Faraco V , Giovanni S ( 2010 ) Heterologous laccase production and its role in industrial applications . Bioeng Bugs 1 : 252 - 62 .
15. Wong KS , Cheung MK , Au CH , Kwan HS ( 2013 ) A novel Lentinula edodes laccase and its comparative enzymology suggest guaiacol-based laccase engineering for bioremediation . PLoS One 8 : e66426 .
16. Bao S , Teng Z , Ding S ( 2013 ) Heterologous expression and characterization of a novel laccase isoenzyme with dyes decolorization potential from Coprinus comatus . Mol Biol Rep 40 : 1927 - 36 .
17. Lu X , Ding SJ ( 2010 ) Effect of Cu2+, Mn2+ and aromatic compounds on the production of laccase isoforms by Coprinus comatus . Mycoscience 51 : 68 - 74 .
18. Hoshida H , Nakao M , Kanazawa H , Kubo K , Hakukawa T , et al. ( 2001 ) Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts . J Biosci Bioeng 92 : 372 - 80 .
19. Tamura K , Dudley J , Nei M , Kumar S ( 2007 ) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4 .0. Mol Biol Evol 24 : 1596 - 1599 .
20. Zheng F , Huang J , Yin Y , Ding S ( 2013 ) A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase . J Ind Microbiol Biotechnol 40 : 1083 - 1093 .
21. Childs RE , Bardsley WG ( 1975 ) The steady-state kinetics of peroxidase with 2,29-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen . Biochem J 145 : 93 - 103 .
22. Canters GW , Gilardi G ( 1993 ) Engineering type 1 copper sites in proteins . FEBS Lett 325 : 39 - 48 .
23. Piontek K , Antorini M , Choinowski T ( 2002 ) Crystal structure of a laccase from the fungus Trametes versicolor at 0.90 Au resolution containing a full complement of coppers . J Biol Chem 277 : 37663 - 37669 .
24. Koikeda S , Ando K , Kaji H , Inoue T , Murao S , et al. ( 1993 ) Molecular cloning of the gene for bilirubin oxidase from Myrothecium verrucaria and its expression in yeast . J Biol Chem 268 : 18801 - 9 .
25. Christenson A , Shleev S , Mano N , Heller A , Gorton L ( 2006 ) Redox potentials of the blue copper sites of bilirubin oxidases . Biochim Biophys Acta 1757 : 1634 - 41 .
26. Uthandi S , Saad B , Humbard MA , Maupin-Furlow JA ( 2010 ) LccA, an archaeal laccase secreted as a highly stable glycoprotein into the extracellular medium by Haloferax volcanii . Appl Environ Microbiol 76 : 733 - 43 .
27. Klonowska A , Gaudin C , Fournel A , Asso M , Le Petit J , et al. ( 2002 ) Characterization of a low redox potential laccase from the basidiomycete C30 . Eur J Biochem 269 : 6119 - 25 .
28. Klonowska A , Gaudin C , Asso M , Fournel A , Reglier M , et al. ( 2005 ) LAC3, a new low redox potential laccase from Trametes sp. strain C30 obtained as a recombinant protein in yeast . Enzyme Microb Technol 36 : 34 - 41 .
29. Jolivalt C , Madzak C , Brault A , Caminade E , Malosse C , et al. ( 2005 ) Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications . Appl Microbiol Biotechnol 66 : 450 - 456 .
30. Guo M , Lu FP , Du LX , Pu J , Bai DQ ( 2006 ) Optimization of the expression of a laccase gene from Trametes versicolor in Pichia methanolica . Appl Microbiol Biotechnol 71 : 848 - 52
31. Brown MA , Zhao Z , Mauk AG ( 2002 ) Expression and characterization of a recombinant multi-copper oxidase: laccase IV from Trametes versicolor . Inorg Chim Acta 331 : 232 - 8 .
32. Cassland P , Jonsson LJ ( 1999 ) Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature . Appl Microbiol Biotechnol 52 : 393 - 400 .
33. Bohlin C , Jonsson LJ , Roth R , van Zyl WH ( 2006 ) Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger . Appl Biochem Biotechnol 129-132 : 195 - 214 .
34. Baker CJO , White TC ( 2000 ) Expression of the laccase IV gene from Trametes versicolor in Trichoderma reesei . Abst Papers Am Chem Soc 219:154
35. Macauley-Patrick S , Fazenda ML , McNeil B , Harvey LM ( 2005 ) Heterologous protein production using the Pichia pastoris expression system . Yeast 22 : 249 - 70 .
36. Bulter T , Alcalde M , Sieber V , Meinhold P , Schlachtbauer C , et al. ( 2003 ) Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution . Appl Environ Microbiol 69 : 987 - 95 .
37. Bai Y , Wang J , Zhang Z , Shi P , Luo H , et al. ( 2010 ) Expression of an extremely acidic beta-1,4-glucanase from thermoacidophilic Alicyclobacillus sp. A4 in Pichia pastoris is improved by truncating the gene sequence . Microb Cell Fact 9 : 33 .
38. Andberg M , Hakulinen N , Auer S , Saloheimo M , Koivula A , et al. ( 2009 ) Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure . FEBS J 276 : 6285 - 300 .
39. Zhao D , Zhang X , Cui D , Zhao M ( 2012 ) Characterisation of a novel white laccase from the deuteromycete fungus Myrothecium verrucaria NF-05 and its decolourisation of dyes . PLoS One 7 ( 6 ): e38817 .
40. Lin Y , Zhang Z , Tian Y , Zhao W , Zhu B , et al. ( 2013 ) Purification and characterization of a novel laccase from Coprinus cinereus and decolorization of different chemically dyes . Mol Biol Rep 40 : 1487 - 1494 .
41. Murugesan K , Yang IH , Kim YM , Jeon JR , Chang YS ( 2009 ) Enhanced transformation of malachite green by laccase of Ganoderma lucidum in the presence of natural phenolic compounds . Appl Microbiol Biotechnol 82 : 341 - 350 .
42. Yan KL , Wang HX , Zhang XY , Yu HB ( 2009 ) Bioprocess of triphenylmethane dyes decolorization by Pleurotus ostreatus BP under solid-state cultivation . J Microbiol Biotechnol 19 : 1421 - 1430 .
43. Ciullini I , Tilli S , Scozzafava A , Briganti F ( 2008 ) Fungal laccase, cellobiose dehydrogenase, and chemical mediators: combined actions for the decolorization of different classes of textile dyes . Bioresour Technol 99 : 7003 - 10 .
44. Kokol V , Doliska A , Eichlerova I , Baldrian P , Nerud F ( 2007 ) Decolorization of textile dyes by whole cultures of Ischnoderma resinosum and by purified laccase and Mn-peroxidase . Enzyme Microb Technol 40 : 1673 - 1677 .